Method for producing recombinant trypsin

ABSTRACT

The present invention concerns a method for producing recombinant trypsin from porcine pancreas in  Pichia pastoris  which is soluble and secreted into the culture medium, whereby expression at pH 3.0-4.0 substantially prevents activation of trypsinogen to β-trypsin and autolysis of β-trypsin by α-trypsin into ε-trypsin and from there into inactive peptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/470,508, filed Apr. 12, 2004, now U.S. Pat. No. 7,276,605, which is aU.S. national counterpart application of international applicationserial No. PCT/EP02/01072, filed Feb. 1, 2002, which claims the benefitof European application No. 01102342.1, filed Feb. 1, 2001.

DESCRIPTION

The invention concerns a method for the recombinant production oftrypsin. For this purpose a trypsinogen with a shortened propeptidesequence is preferably expressed in a recombinant host cell and secretedinto the culture medium in an uncleaved form. Subsequently thepropeptide sequence is cleaved in a controlled manner to form activetrypsin.

Trypsin is a serine protease which catalyses a hydrolytic cleavage ofpeptides at the carboxyl group of the basic amino acids arginine andlysine (Keil B., 1971). Trypsin from bovine pancreas was one of thefirst proteolytic enzymes that could be used in a pure form and inadequate quantities for exact chemical and enzymatic studies (Northropet al., 1948). It was subsequently also possible to isolate proteasesthat can be allocated to the trypsin family from other highervertebrates (pig—Charles et al. 1963/sheep—Bricteux-Gregoire et al.(1966); Travis (1968)/turkey —Ryan (1965)/humans—Travis et al. (1969)and others). At this time the first enzymes belonging to the trypsinfamily were also isolated from two species of streptomyces (Morihara andTsuzuki (1968); Trop and Birk (1968); Wählby and Engström (1968); Wählby(1968; Jurasek et al. (1969)).

The enzyme is synthesized in the pancreas cells of vertebrates as aninactive precursor, trypsinogen, and subsequently converted into theactive form by cleavage of the propeptide (Northrop et al. (1948),Desnuelle (1959)). The first trypsinogen molecules were activatednaturally by the enteropeptidase enterokinase which hydrolyses thepeptide bond between (Asp4)-Lys-⇓-Ile (SEQ ID NO. 25) which cleaves offthe propeptide (Keil (1971)). The recognition sequence of theenterokinase (Asp4)-Lys (SEQ ID NO. 24) is accordingly located directlyat the C-terminus of the propeptide in almost all previously knowntrypsinogen molecules (Light et al. (1980)). The activation process canalso proceed autocatalytically at physiological pH values since a lysineis located on the C-terminal side of the enterokinase recognitionsequence and hence the Lys-⇓-Ile peptide bond can also be hydrolyzed bytrypsin (Light et al. (1980)).

Trypsin has always been an interesting protease for biotechnologicalapplications due to its ready availability from various mammals, highspecificity (only cleaves at the C-terminal side of lysine or arginine)together with high specific activity (˜150 U/mg) and its good storagestability. Trypsin is mainly used for the tryptic cleavage of peptidesinto small sections for sequencing, for detaching adherent cells fromcoated cell culture dishes and for cleaving fusion proteins into thetarget peptide and the fusion component, for activating propeptides(e.g. trypsinogen to trypsin) and for the recombinant production ofpeptide hormones (e.g. proinsulin to insulin, cf. WO 99/10503). Trypsinis also a component of some pharmaceutical preparations (ointments,dragees and aerosols for inhalation (“Rote Liste”, 1997; The UnitedStates Pharmacopeia, The National Formulary, USP23-NF18, 1995)). Sincethe use of enzymes from animal sources is no longer permitted in manycases (potential contamination with infectious agents), recombinanttrypsin molecules for the desired biotechnological applications have tobe provided from microbial hosts.

There are several methods for the recombinant production of trypsin fromvarious organisms.

Graf, L. et al (1987 and 1988) describe the expression and secretion ofrat trypsin and trypsinogen mutants in E. coli. In order to secrete thetrypsinogen molecules into the periplasm of E. coli the nativetrypsinogen signal sequence is replaced by the signal sequence of thebacterial alkaline phosphatase (phoA). The secreted inactive trypsinogenmolecules are isolated from the periplasm and activated by enzymaticcleavage using purified enterokinase.

Vasquez, J. R. et al. (1989) describe the expression and secretion ofanionic rat trypsin and trypsin mutants in E. coli. In order to expressand secrete the active trypsin molecules into the periplasm of E. coli,the native trypsinogen prepro segment (signal sequence and activationpeptide) is replaced by the signal sequence of the bacterial alkalinephosphatase (phoA) and the phoA promoter that can be regulated byphosphate is used. Active trypsin is isolated from the periplasm.However, the yield is very low (ca. 1 mg/l).

Higaki, J. N. et al. (1989) describe the expression and secretion oftrypsin and trypsin mutants into the periplasm of E. coli using the tacpromoter and the S.typhimurium hisJ signal sequence. The yield of activetrypsin is ca. 0.3 mg/l. The volume yield of active anionic rat trypsincan be increased to about 50 mg/l by high cell density fermentation(Yee, L. and Blanch, H. W., (1993)). However, the authors refer toproblems in the expression and secretion of active trypsin in E. coli.Enzymatically active trypsin is formed in the periplasm of E. coli aftercleavage of the signal sequence and native trypsin protein folding toform 6 disulfide bridges. The formation of active trypsin is toxic forthe cell since active trypsin hydrolyses the periplasmatic E. coliproteins which lyses the cells. Moreover the protein folding of trypsinand in particular the correct native formation of the 6 disulfidebridges appears to be impeded in the periplasm of E. coli. The system isnot suitable for the isolation of relatively large amounts of trypsin(>10 mg; Willett, W. S. et al., (1995)).

In order to produce larger amounts of trypsin (50-100 mg) for X-raycrystallographic investigations, an inactive trypsinogen precursor isproduced in yeast under the control of a regulatable ADH/GAPDH promoterand secreted by fusion with the yeast α factor leader sequence. Theexpression product secreted into the medium is converted quantitativelyinto trypsin in vitro by means of enterokinase. The yield is 10-15 mg/l(Hedstrom, L. et al. (1992)).

DNA sequences are described in EP 0 597 681 which code for mature bovinetrypsin and bovine trypsinogen with an initial methionine residue. Inaddition the expression in E. coli is described but the strategy of howactive trypsin is formed in E. coli is not explained.

A method for producing trypsin from porcine pancreas or a derivativethereof in Aspergillus by a recombinant method is described in WO97/00316. A vector is used for transformation which codes fortrypsinogen or a derivative thereof which is fused at the N-terminus toa functional signal peptide. However, yeast cultures achieve higherbiomass concentrations compared to Aspergillus cultures and growconsiderably more rapidly and thus the specific expression yield peryeast cell can be less than that for Aspergillus cells in order toachieve yields that are required for an economic expression method.

A method for the recombinant production of a zymogenic precursor ofproteases which contains an autocatalytic cleavage site that does notoccur naturally is described in WO 99/10503. The method comprises anexpression of the zymogenic precursor in E. coli in the form ofinclusion bodies with subsequent purification and renaturation underconditions where the protease part of the zymogenic precursor is formedin its natural conformation and the cleavage of the renatured zymogenicprecursor then occurs autocatalytically. A disadvantage of this methodare the large losses that often occur during renaturation and the largevolumes that are required for an industrial production.

The recombinant production of trypsin analogues in Pichia pastoris isdescribed in WO 00/17332. A vector is used for the transformation whichcodes for a trypsinogen analogue (derivative of bovine trypsinogen) inwhich the amino acid lysine at the C-terminus of the propeptide wasexchanged by mutation for another amino acid (apart from arginine orlysine) and which is fused N-terminally to a functional signal peptide.In this method the trypsin analogues are secreted into the medium in asoluble form and as a result of the incorporated mutation are also notactivated and further degraded by undesired autocatalysis even atrelatively high pH values of the fermentation process. An aminopeptidaseis then used for activation. However, a disadvantage of this method isthe need to remove the additional aminopeptidase which may havedisadvantageous side activities for the subsequent use of the trypsin inthe final process.

The expression of naturally occurring trypsinogen genes in Pichiapastoris is described in WO 01/55429. In order to avoid autocatalyticactivation the fermentation is carried out in the example in the lessacid pH range at ca. 6. It is thus outside the optimal pH range whichprevents autocatalytic activation during longer run times in theexpression phase. Other disadvantages of the method are the trypsinogengenes that are not codon-optimized for expression in Pichia pastoris andthe associated low expression yields.

The object of the invention is to provide a method for the recombinantproduction of trypsin in which the disadvantages of the prior art are atleast partially eliminated and which allows active trypsin to beobtained in a simple manner in a high yield and activity. In particularit should be possible to isolate trypsinogen as an intermediate productin a soluble form from the culture medium of the expression host whichshould not be subject to any substantial degree of autocatalyticactivation during the fermentation and purification. Furthermore itshould be possible to subsequently activate the trypsinogenautocatalytically and/or by adding recombinant trypsin.

The object according to the invention is achieved by a method for therecombinant production of trypsin comprising the steps:

a) transforming a host cell with a recombinant nucleic acid which codesfor a trypsinogen with an enterokinase recognition site in thepropeptide sequence in a secretable form, preferably fused with a signalpeptide that mediates secretion,

b) culturing the host cell under conditions which enable an expressionof the recombinant nucleic acid and a secretion of the expressionproduct into the culture medium, the conditions being selected such thatan autocatalytic cleavage of the propeptide sequence is at leastsubstantially prevented,

c) isolating the expression product from the culture medium,

d) cleaving the propeptide sequence to form active trypsin and

e) optionally separating non-cleaved trypsinogen molecules from activetrypsin.

The host cell used in the method according to the invention can be aeukaryotic or prokaryotic host cell like those known from the prior art.The host cell is preferably a eukaryotic cell, particularly preferably afungal cell and most preferably a yeast cell. Suitable examples of yeastcells are Pichia pastoris, Hansenula polymorpha, Saccharomycescerevisiae, Schizosaccharomyces pombae where methylotrophic yeasts suchas Pichia pastoris or Hansenula polymorpha and in particular Pichiapastoris are preferably used.

It is expedient to use a host cell that is able to express therecombinant nucleic acid and to secrete the expression product into theculture medium under conditions where an autocatalytic cleavage of thepropeptide sequence is at least substantially and preferably essentiallyquantitatively prevented. Such conditions advantageously comprise anacidic pH value of the culture medium, particularly preferably a pHvalue in the range between 3 and 4. Trypsinogen is stable at acidic pHvalues and especially at pH values of up to 4 whereas trypsin isautocatalytically activated in a neutral or alkaline medium (Keil, B.(1971)).

The culture under suitable conditions that occurs in the methodaccording to the invention prevents a premature activation of therecombinant trypsinogen secreted by the host cell which can alsosubstantially prevent further degradation of the resulting trypsin to asfar as inactive peptides. The stability of the recombinant trypsinogenunder the culture conditions is important since the expression should beaccompanied by a secretion. In this connection secretion from the hostcell is understood as the discharge of trypsinogen from the cytoplasmthrough the cell membrane into the culture medium. This usually occursby N-terminal fusion of the trypsinogen with a functional signalpeptide. Examples of suitable signal peptides are known signal peptidesfrom yeast and especially preferably the signal peptide of the a factorfrom Saccharomyces cerevisiae. However, it is of course also possible touse other signal peptides e.g. the signal peptide which naturallycontrols the secretion of trypsinogen.

In order to improve the expression yield of the recombinant trypsin, arecombinant nucleic acid with an optimized codon usage for therespective host cell is preferably used. Accordingly it is advantageousto use a nucleic acid for a yeast cell which has an optimized codonusage for yeast. Such nucleic acids having an optimized codon usage canfor example be produced by synthesizing individual oligonucleotides inparallel and subsequently combining them.

The method according to the invention is basically suitable forproducing any types of recombinant trypsin provided the respectivetrypsinogen that is secreted as an expression product by the host cellis essentially stable under the culture conditions. The method ispreferably used to produce trypsin from vertebrates, in particular frommammals such as pigs, sheep or humans. Porcine trypsin is particularlypreferably produced.

Furthermore it is preferred that a recombinant nucleic acid is usedwhich codes for a trypsinogen with a shortened propeptide sequence.Trypsinogen is understood as a protein which, although it has formed asubstantially correct protein structure, has no or only a very lowproteolytic activity compared to active trypsin which is advantageouslyat least 5-fold and particularly preferably at least 10-fold less thanthe proteolytic activity of the active form. The natural length of thepropeptide part, e.g. 25 amino acids in the case of a porcinetrypsinogen, is shortened preferably down to a propeptide sequence whichonly consists of a recognition sequence for an enteropeptidase e.g.enterokinase e.g. an amino acid sequence Asp-Asp-Asp-Asp-Lys (SEO ID NO.24). A few additional amino acids that are due to the cloning, e.g. upto 5 amino acids, can be optionally attached to the N-terminus of theenterokinase recognition sequence.

It was found that the expression of a shortened recombinant trypsinogenaccording to the invention containing the amino acids Glu-Phe attacheddue to the cloning to the N-terminus of the enterokinase recognitionsite results in significantly higher yields than is the case for theexpression of natural trypsinogen in the method according to theinvention. Hence in a particularly preferred embodiment of the methodaccording to the invention a recombinant nucleic acid having thenucleotide sequence shown in SEQ ID NO.22 is used which codes for aporcine trypsinogen with a shortened propeptide sequence.

In the method according to the invention the expression of therecombinant trypsinogen is preferably controlled by an inducibleexpression control sequence. Hence the growth of the host cell can takeplace before induction of expression under conditions which arefavourable or optimal for the growth of the host cell. Thus for examplegrowth can take place at pH 5-7, in particular pH 5-6 up to apredetermined optical density during which the regulatable expressioncontrol sequence is repressed. Expression can then be induced bychanging the temperature and/or by adding an inducer. An example of apreferred expression control sequence is the AOX 1—promoter from Pichiapastoris that can be induced by methanol which is particularly suitablefor inducible expression in methylotrophic yeasts. The cultureconditions are advantageously changed before induction of the expressioncontrol sequence in such a manner that for example by changing the pH toa range of 3-4 the trypsinogen formed by expression of the recombinantnucleic acid accumulates in an intact form in the culture medium.

The culture conditions in the method according to the invention areselected such that autocatalytic cleavage of the propeptide sequence issubstantially prevented. After isolating the expression product from theculture medium, the propeptide sequence can be cleaved off undercontrolled conditions. This cleavage can for example be carried out byadding recombinant trypsin or/and by autocatalytic cleavage. In thisconnection autocatalytic cleavage is understood as the self-activationof recombinant trypsinogen which may be optionally accelerated by addingsmall amounts of recombinant trypsin but without adding a foreignprotein. In this manner it is not necessary to use an additional foreignprotein which is usually derived from animal raw material sources andhas to be subsequently removed again which could cause undesiredcleavages in the subsequent application. The autocatalytic activationpreferably occurs at a pH in the range of 7-8. This ensures that thetrypsinogen formed during the expression can be firstly highly purifiedin a strongly acidic range and the activation can be specificallystarted by rebuffering preferably in the presence of small amounts e.g.20 mM CaCl₂ to a neutral to weakly basic pH range. The activation can bestopped by changing the pH again to a strongly acidic range. Trypsin canfor example be purified by chromatography on ion exchanger material suchas SP-Sepharose®XL or benzamidine-Sepharose.

In order to purify the expression product from the culture medium, theculture supernatant can be firstly separated from the cells as describedin example 6.1. The subsequent purification comprising an autocatalyticactivation of the trypsinogen is preferably carried out by suitablechromatographic purification procedures. In a particularly preferredembodiment a chromatography is carried out as described in example 6.2.without prior separation of the cells in which a buffer containingcalcium ions preferably at a final concentration of 1-30 mM calcium isadded to the culture medium which still contains the cells. Subsequentlysteps (c), (d) and optionally (e) of the method according to theinvention are carried out for example using suitable chromatographicpurification procedures and a rebuffering step for the autocatalyticcleavage of the expression product.

Hence a particularly preferred embodiment of the method according to theinvention is characterized by:

a) transforming a host cell with a recombinant nucleic acid which codesfor the zymogenic precursor trypsinogen from the pig which is fused witha signal peptide sequence and contains a propeptide which is shorteneddown to the enterokinase recognition sequence, where this cleavage siteis cleaved by recombinant trypsin or is autocatalytically cleaved undercertain buffer conditions and the shortened trypsinogen can be cleavedto form active trypsin in this process,

b) culturing the host cell during the expression phase at an acidic pH,preferably pH 3-4 so that the shortened trypsinogen is present in asoluble form and secreted into the culture medium but the autocatalyticactivation is substantially prevented,

c) isolating the shortened recombinant trypsinogen from the culturesupernatant and activating it under conditions which allow an effectivecleavage of the shortened trypsinogen by recombinant trypsin or byautocatalytic cleavage and

d) optionally separating non-cleaved trypsinogen molecules from activetrypsin.

The shortening of the propeptide described in this invention has apositive effect especially on expression and autocatalytic activation.Furthermore after the autocatalytic activation, the undesired furtherautolysis increases significantly at a pH of >4.0 during the growthphase and expression phase of the culture.

An additional increase in the expression yield in the method accordingto the invention can be achieved by transforming the host cell withseveral vectors each of which contains a recombinant nucleic acid asstated above where the vectors contain different selection markers e.g.Zeocin and G418. Culture under multiple selection conditionssurprisingly allows the expression yield of recombinant trypsinogen tobe increased considerably further.

Another subject matter of the invention is a recombinant nucleic acidwhich codes for a trypsinogen having an enterokinase recognition site inthe propeptide sequence where the propeptide sequence is shortenedrelative to the natural propeptide sequence and is preferably fused witha signal peptide sequence. The shortened propeptide sequence accordingto the invention preferably consists of an enterokinase recognition sitehaving the amino acid sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO. 24) andoptionally up to 5 additional amino acids located arranged N-terminallythereof. The nucleic acid according to the invention particularlypreferably has the nucleotide sequence shown in SEQ ID NO.22.

The nucleic acid according to the invention is preferably in operativelinkage with a regulatable expression control sequence which is forexample a suitable expression control sequence for gene expression inyeast cells such as the AOX1 promoter from Pichia pastoris.

The invention also concerns a recombinant vector which contains at leastone copy of a recombinant nucleic acid as stated above. The vector ispreferably a vector that is suitable for gene expression in yeast cells.Examples of such vectors are described in Sambrook et al., Molecularcloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

In addition to the recombinant nucleic acid, the vector contains othersuitable genetic elements for the respective intended use and inparticular a selection marker gene. Vectors are particularly preferablyused which can be present in the cell in multiple copies especiallyvectors which can be integrated in a multiple form into the genome ofthe host cell. The invention also concerns combinations of vectors whicheach contain different selection marker genes and which can bepropagated concurrently in a host cell.

In addition the invention concerns a recombinant cell which istransformed with a nucleic acid according to the invention or a vectoraccording to the invention. The recombinant cell is preferably a yeastcell in particular a methylotrophic yeast cell such as Pichia pastorisor Hansenula polymorpha.

Finally the invention concerns a recombinant trypsinogen which is codedby a nucleic acid according to the invention. The recombinanttrypsinogen has a propeptide sequence which is shortened compared to thenatural propeptide sequence and contains an enterokinase recognitionsite. The recombinant trypsinogen according to the invention preferablyhas the amino acid sequence shown in SEQ ID NO. 23.

The present invention is also elucidated by the following figures andexamples.

FIG. 1: shows a plasmid map of the expression plasmid pTRYP-9 containingthe complete recombinant trypsinogen,

FIG. 2: shows a plasmid map of the expression plasmid pTRYP-11containing the shortened recombinant trypsinogen (sh-trypsinogen) andthe Zeocin resistance marker gene (ZeoR),

FIG. 3: shows a plasmid map of the expression plasmid pTRYP-13containing the shortened recombinant trypsinogen (sh-trypsinogen) and akanamycin/G418 selection marker gene (KanR).

EXAMPLES

Methods:

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described by Sambrook,J. et al. (1989) in Molecular Cloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Protein Determination

The protein determination of purified trypsin was carried out bymeasuring the absorbance at 280 nm. A value of 13.6 was used for A1%/280 nm for 1 cm path length.

Calculation:Protein [mg/ml]=(10 [mg/ml]*ΔA _(sample)*dilution)/13.6Pichia pastoris and Expression Vectors for Pichia pastoris

The catalogue and instruction manuals from Invitrogen were used asinstructions for handling Pichia pastoris and the expression vectors.The vectors for expressing the shortened recombinant trypsinogen arebased on the vectors pPICZαA and pPIC9K from Invitrogen.

Example 1

Gene synthesis of the complete recombinant trypsinogen with optimizedcodon usage for expression in yeast

One of the preferred methods for providing the method according to theinvention is to synthesize a codon-optimized gene sequence. In order tooptimize each codon for expression in yeast it was necessary to carryout a complete de novo synthesis of the ca. 700 bp gene which codes forthe complete recombinant trypsinogen. It was possible to optimize eachcodon when necessary by utilizing the degenerate code in theretranslation of the amino acid sequence of porcine trypsinogenaccording to SEQ ID NO.1. For this purpose the gene was divided into 18oligonucleotides having a length of 54 to 90 nucleotides. Theoligonucleotides were designed as an alternating sequence of sensestrand and counter-strand fragments whose 5′ and 3′ ends each overlappedin a complementary manner with the neighboring oligonucleotides. Theoverlapping region was selected in each case such that unspecificbinding was largely prevented in the annealing reaction in thesubsequent PCR reaction. The oligonucleotides at the 5′ and 3′ end ofthe gene were provided upstream and downstream of the coding region withrecognition sites for restriction endonucleases which could be used forthe later insertion of the synthetic gene according to SEQ ID NO.2 intoexpression vectors. Thus a recognition site for the restrictionendonuclease EcoRI was incorporated upstream and a recognition site forthe restriction endonuclease XbaI was incorporated downstream. Thesequences of the oligonucleotides are shown in SEQ ID NO.3 to 20.

Gene synthesis was carried out by means of the PCR reaction. For thisthe coding region was firstly divided into three segments(oligonucleotides 3 to 8, 9 to 14, 15 to 20) and these segments weregenerated in separate PCR reactions using overlapping complementaryoligonucleotides. In this process the gene fragment was extended in astepwise manner till the full length product was formed which was thenamplified in subsequent cycles. The annealing temperature was selectedaccording to the overlapping region with the lowest melting temperature.

The three segments were subsequently analysed by agarose gelelectrophoresis, the products having the expected length were isolatedfrom the gel by means of the QIAquick Gel Extraction Kit (Qiagen) andsynthesized to form the complete gene product in a further PCR reaction.The first 5 cycles of the PCR reaction were carried out without addingthe primer at the 5′ end and at the 3′ end of the entire gene so that atfirst a few fragments of the gene product of the expected length wereformed from the three segments. The annealing temperature was selectedaccording to the overlapping region with the lowest melting temperature.Then the terminal primers were added and the annealing temperature wasincreased according to the annealing temperature of the primer with thelowest melting temperature. The gene fragment of the expected length wasthen amplified in a further 25 cycles. The PCR fragment was checked bysequencing.

Example 2

Generation of the Shortened Trypsinogen Gene

The codons for the first 20 amino acids of the naturally occurringtrypsinogen were deleted by means of a specially designed 5′ primeraccording to SEQ ID NO. 21 such that only the codons for the recognitionsequence of the enteropeptidase enterokinase Asp-Asp-Asp-Asp-Lys (SEO IDNO. 24) and the codons Glu-Phe necessary for cloning into the expressionvectors for Pichia pastoris due to the EcoRI restriction endonucleaserecognition sequence remain as the sequence of the propeptide at theN-terminus of the shortened recombinant trypsinogen. The deletion wasintroduced by a PCR reaction on the PCR fragment of the gene for thecomplete recombinant trypsinogen using the 5′ primer according to SEQ IDNO. 21 and the 3′ primer according to SEQ ID NO. 20. The DNA sequenceand the amino acid sequence of the shortened recombinant trypsinogen areshown in SEQ ID NO. 22 and SEQ ID NO. 23 respectively.

Example 3

Cloning the PCR Fragments of the Complete Recombinant TrypsinogenGenerated by Gene Synthesis and of the Shortened Recombinant Trypsinogeninto Expression Vectors for Pichia pastoris

The PCR fragments were recleaved with EcoRI and XbaI (Roche DiagnosticsGmbH), isolated again (QIAquick Gel Extraction Kit/Qiagen) andsubsequently ligated into a fragment of the expression vector pPICZαA(Invitrogen) linearized with EcoRI and XbaI (Roche Diagnostics GmbH) andisolated with the QIAquick Gel Extraction Kit/Qiagen. For this 1 μl (20ng) vector fragment and 3 μl (100 ng) PCR fragment, 1 μl 10× ligasebuffer (Sambrook et al., 1989 B.27), 1 μl T4 DNA ligase, 4 μl sterileH₂O_(redistilled) were pipetted, carefully mixed and incubated overnightat 16° C. In this vector the synthetic gene is under the control of theAOX 1 promoter (promoter for the alcohol oxidase 1 from Pichia pastoris)that can be induced with methanol (Mallinckrodt Baker B. V.) and islocated in the correct reading frame behind the signal peptide of the afactor from Saccharomyces cerevisiae. In order to check this and isolatethe plasmid, 5 μl of the ligation mixture were then transformed in 200μl competent cells (Hanahan (1983) of E. coli XL1Blue (Stratagene). A 30min incubation on ice was followed by a heat shock (90 sec at 42° C.).Subsequently the cells were transferred to 1 ml LB medium and incubatedfor 1 hour at 37° C. in LB medium for phenotypic expression. Aliquots ofthis transformation mixture were plated out on LB plates using 100 μg/mlZeocin as the selection marker and incubated for 15 hours at 37° C. Theplasmids were isolated from the grown clones (Sambrook, J. et al. (1989)In. Molecular cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and then checked for anerror-free base sequence by means of restriction analysis andsequencing. The expression vectors formed in this manner which contain asynthetic gene for the complete recombinant trypsinogen or a shortenedrecombinant trypsinogen were named pTRYP-9 (see FIG. 1) and pTRYP-11(see FIG. 2).

Transformation of pTRYP-9 and pTRYP-11 into Pichia pastoris

For the transformation of pTRYP-9 and pTRYP-11 into Pichia pastorisX-33, GS 115 or KM71H with subsequent integration into the genome, thevector was firstly linearized with SacI (Roche Diagnostics GmbH). Thetransformation was carried out by electroporation using the Gene PulserII (Biorad). For this a colony of Pichia pastoris was inoculated with 5ml YPD medium (Invitrogen) and incubated overnight at 30° C. whileshaking. The overnight culture was subsequently reinoculated 1:2000 in200 ml fresh YPD medium (Invitrogen) and incubated overnight at 30° C.while shaking until an OD₆₀₀ of 1.3 to 1.5 was reached. The cells werecentrifuged (1500×g/5 minutes) and the pellet was resuspended in 200 mlice-cold, sterile water (0° C.). The cells were again centrifuged(1500×g/5 minutes) and resuspended in 100 ml ice-cold, sterile water (0°C.). The cells were again centrifuged and resuspended in 10 ml ice-cold(0° C.) 1 M sorbitol (ICN). The cells were again centrifuged andresuspended in 0.5 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cellsisolated in this manner were kept on ice and immediately used for thetransformation.

80 μl of the cells were admixed with ca. 1 μg linearized pTRYP-9 orpTRYP-11 vector DNA and the entire mixture was transferred into anice-cold (0° C.) electroporation cuvette and incubated for a further 5minutes on ice. Subsequently the cuvette was placed in the Gene PulserII (Biorad) and transformation was carried out at 1 kV, 1 kΩ and 25 μF.After electroporation the mixture was admixed with 1 ml 1 M sorbitol(ICN) and subsequently 100 to 150 μl was plated out on a YPDS agar plate(Invitrogen) containing 100 μg/ml Zeocin (Invitrogen). The plates weresubsequently incubated for 2-4 days at 30° C.

The clones were reinoculated on raster MD (=minimal dextrose) plates andanalysed further. Grown clones were picked, resuspended in 20 μl sterilewater, lysed (1 hour, 30° C., 10 min, −80° C.) with 17.5 U lyticase(Roche Diagnostics GmbH) and directly examined for correct integrationof the synthetic trypsinogen expression cassette by means of PCR.

Clones which had integrated the complete expression cassette duringtransformation into the genome, were then used in expressionexperiments.

Expression of the Complete and Shortened Recombinant Trypsinogen

Positive clones were inoculated in 3 ml BMGY medium (Invitrogen) andincubated overnight at 30° C. while shaking. Subsequently the OD wasdetermined at 600 nm and they were reinoculated in 10 ml BMMY medium(Invitrogen) pH 3.0 such that the resulting OD₆₀₀ was 1. The BMMY medium(Invitrogen) pH 3.0 contains methanol (Mallinckrodt Baker B. V.) whichinduces the expression of the complete or shortened recombinanttrypsinogen via the AOX1 promoter.

The shaking flasks were incubated at 30° C. while shaking, samples weretaken every 24 hours, the OD₆₀₀ was determined, an aliquot was taken tocheck the expression of the complete and shortened recombinanttrypsinogen by means of SDS polyacrylamide gel electrophoresis and eachwas supplemented with 0.5% methanol (Mallinckrodt Baker B. V.) forfurther induction. The expression experiments were carried out for 72hours.

Analysis of the Expression of the Complete and Shortened RecombinantTrypsinogen by Means of SDS Gel Electrophoresis

500 μl were taken from each expression culture, the OD₆₀₀ was determinedand the cells were centrifuged. The culture supernatant was stored andthe cell pellet was resuspended for the lysis in an appropriate amountof Y-PER™ (50 to 300 μl/Pierce) for the OD₆₀₀ and shaken for 1 hour atroom temperature. Subsequently the lysate was centrifuged to separatecell debris (15000×g/5 minutes) and the supernatant was transferred tofresh reaction vessels. 10 μl lysate and 10 μl of the culturesupernatant were applied to an SDS polyacrylamide gel and the proteinswere separated according to size by applying an electrical field.

Surprisingly it was possible to identify weak bands in the culturesupernatants of clones containing the complete recombinant trypsinogenas well as of clones containing the shortened recombinant trypsinogenthat did not occur in the control clones. Control clones are to beunderstood as Pichia pastoris X33 cells that have been transformed withthe starting vector pPICZαA and that have been grown and inducedanalogously to the expression clones for trypsinogen. The migrationproperties of the new protein bands in the expression clones correspondsto the calculated molecular weight and is slightly higher than that ofbovine trypsin that had been applied as the control marker. This slightdifference in size indicates an intact, non-activated trypsinogen.

Surprisingly the recombinant shortened trypsinogen was expressedsignificantly more strongly than the recombinant complete trypsinogen.

Example 4

Increasing the Expression Yield by Multiple Transformation

The best clones from the expression experiments with the recombinantshortened trypsinogen were again prepared for electroporation asdescribed above and again transformed with 1 μg linearized pTRYP-11vector DNA and the transformation mixture was plated out on YPDS agarplates (Invitrogen) containing 1000 to 2000 μg/ml Zeocin (Invitrogen).This increases the selection pressure in such a way that only clones cangrow that have integrated several copies of the expression vectorpTRYP-11 into the genome and hence also several copies of the respectiveresistance gene (in this case Zeocin®). The Zeocin® resistance proteinis the product of the bleomycin gene from Streptoalloteichus hindustanus(Calmels, T. et al., (1991); Drocourt, D. et al., (1990)), which bindsZeocin® in a stoichiometric concentration ratio and hence makes the cellresistant to Zeocin®. The higher the concentration of Zeocin® in theYPDS agar plates, the more resistance protein has to be produced by thecell in order to quantitatively bind Zeocin® and thus allow growth. Thisis among others possible when multiple copies of the resistance gene areintegrated into the genome. Clones were reinoculated on raster MD platesas described above. Subsequently these clones were in turn checked fortrypsinogen expression and secretion as described above.

Surprisingly it was possible after this measure to identify cloneshaving considerably increased expression yield of the shortenedrecombinant trypsinogen secreted into the culture supernatant after SDSpolyacrylamide gel electrophoresis.

Example 5

Increasing the Expression Yield by Using a Second Selection Pressure

Increasing the Zeocin® concentration above 2000 μg/ml did not lead to animproved expression yield of the shortened recombinant trypsinogen. Inorder to further increase the gene copy number in the expression clonesof the gene according to SEQ ID NO. 22 which codes for the recombinantshortened trypsinogen and is codon-optimized for expression in yeast,additional expression vectors were integrated into the genome of theexpression clones prepared in examples 3 and 4 having the highestexpression yield by means of a second selection pressure, preferablyG418 (Roche Diagnostics GmbH).

For this purpose a part of the expression cassette from pTRYP-11consisting of a part of the AOX1 promoter, the gene for the signalpeptide of the α factor of Saccharomyces cerevisiae, the codon-optimizedgene for the recombinant shortened trypsinogen according to SEQ IDNO.22, is cut out with the restriction endonucleases SacI and XbaI frompTRYP-11, the restriction mixture is separated on a 1% agarose gel andthe 1693 bp fragment is isolated from the gel (QIAquick Gel ExtractionKit/Qiagen). At the same time the vector pPIC9K (Invitrogen) was cleavedwith SacI and NotI, the restriction mixture was separated on a 1%agarose gel and the 8956 bp vector fragment was isolated from the gel(QIAquick Gel Extraction Kit/Qiagen). The XbaI overhang of the fragmentfrom pTRYP-11 and the NotI overhang from pPIC9K was filled up withKlenow polymerase (Roche Diagnostics GmbH) to form blunt ends accordingto the manufacturer's instructions. Subsequently the two fragmentsobtained in this manner were ligated as described above. The ligationmixture was transformed in E. coli XL1 Blue (Stratagene) as describedabove (the clones containing plasmid were selected by 100 μg/mlampicillin in the nutrient plates) and checked by means of restrictionanalysis and sequencing. The expression vector formed in this way wasnamed pTRYP-13 (see FIG. 3).

The integration of the expression vector pTRYP-13 into the genome ofPichia pastoris was selected by means of G418 (Roche Diagnostics GmbH).

The clones having the highest trypsinogen expression yield from themultiple transformation with pTRYP-11 (Zeocin resistance) were preparedfor electroporation as described above and transformed with 1 μg of thevector fragment from pTRYP-13 (G418 resistance) linearized with SalI(Roche Diagnostics GmbH). The transformation mixture was subsequentlystored for 1 to 3 days at 4° C. in 1 M sorbitol (ICN) (for the formationof G418 resistance), then 100 to 200 μl was plated out on YPD plates(Invitrogen) containing 1, 2 and 4 mg/ml G418 (Roche Diagnostics GmbH)and incubated for 3 to 5 days at 30° C. The clones resulting therefrompreferably from the YPD plates with the highest G418 concentration, wereagain examined for an increased expression of the shortened recombinanttrypsinogen using SDS polyacrylamide gel electrophoresis as describedabove.

After this process is was surprisingly possible to again identify cloneshaving an increased expression yield of the shortened recombinanttrypsinogen in the culture supernatant after SDS polyacrylamide gelelectrophoresis.

Example 6

Isolating Trypsinogen From the Culture Supernatant and Activation 6.1

The culture supernatant was separated from the cells by microfiltration,centrifugation or filtration. The trypsinogen was purified bychromatography on phenyl Sepharose fast flow (Pharmacia). Thechromatography was carried out in a pH range of 2-4. Autocatalyticactivation was started by rebuffering the pH to 7-8 in the presence of20 mM CaCl₂. This autocatalytic activation can be terminated again bychanging the pH back into the range of 2-4. Active trypsin was purifiedby chromatography on benzamidine Sepharose (e.g. SP-Sepharose®XL, ff)(Pharmacia/package insert) or on an ion exchanger. Trypsin is stored atpH 1.5-3 in order to avoid autolysis. The specific activity of thepurified trypsin is 180-200 U/mg protein.

6.2

The entire fermentation broth is diluted in a ratio of about 1:2 to 1:4with ammonium acetate buffer (5-20 mM) containing 5-30 mM calciumchloride, pH 3.5 and purified by means of an expanded bed chromatography(McCornick (1993); EP 0 699 687) using a cation exchanger (e.g.Streamline®SP, XL). In this case the chromatography is carried out inthe presence of the cells. The cells are simultaneously separated by thechromatography step. Subsequently the procedure is as described inexample 6.1 (rebuffering/activation etc.).

Example 7

Activity Determination

The activity of trypsin was determined using Chromozym TRY (PentapharmLtd) in 100 mM Tris pH 8.0, 20 mM CaCl₂ at 25° C. The photometricmeasurement is carried out at 405 nm.

Abbreviations:

YPD: yeast peptone dextrose YPDS: yeast peptone dextrose sorbitol BMGY:buffered glycerol-complex medium BMMY: buffered methanol-complex mediumSDS: sodium dodecyl sulfate

LIST OF LITERATURE REFERENCES

-   Bricteuz-Gregoire, Schyns R., Florkin M (1966) Biochim. Biophys.    Acta 127: pp 277.-   Calmels T., Parriche M., Durand H., Tiraby G. (1991), Curr. Genet.    20: pp 309.-   Charles M., Rovery M., Guidoni A., Desnuelle P. (1963) Biochim.    Biophys. Acta 69: pp 115-129.-   Desnuelle P. (1959) The Enzymes 2^(nd) Edition vol 4 Editor Boyer,    Acad. Press NY. pp. 119.-   Drocourt, D., Calmels T., Reynes J. P., Baron M., Tiraby G. (1990),    Nucleic Acid Research 18: pp 4009.-   Graf L., Craik C. S., Patthy A., Roczniak S., Fletterick R J.,    Rutter W. J. (1987) Biochem. 26: pp. 2616.-   Graf L., Jancso A., Szilagyi L., Hegyi G., Pinter K., Naray-Szabo    G., Hepp J., Mehzihradszky K., Rutter W. J. (1988) Proc. Natl. Acad.    Sci USA 85, pp 4961.-   Hanahan (1983) J. Mol. Biol., 166: pp 557.-   Higaki J. N., Evnin L. B., Craik C. S. (1989) Biochem. 28: pp 9256.-   Hedstrom L., Szilagyi L., Rutter W. J. (1992) Science 255: pp 1249.-   Jurasek L., Fackre D. Smillie L. B. (1969) Biochem. Biophys. Res.    Commun 37: pp. 99.-   Keil B. (1971) The Enzymes Vol II, 3^(rd) Edition, Editor Boyer,    Acad. Press N.Y. pp 249-275.-   Light A., Savithari H. S., Liepnieks J. J. (1980) Analytical    Biochemistry 106: pp 199-206.-   McComick, D. K., Bio/Technol. 11 (1993), 1059; Expanded Bed    Absorption, Principles and Methods, Amersham Pharmacia Biotech,    Edition AB, ISBN 91-630-5519-8.-   Morihara K. and Tszzuki J. (1969) Arch Biochem Biophys 126: pp 971.-   Northrop J. H., Kunitz M., Herriott R. (1948) Crystalline Enzymes,    2^(nd) Edition, Columbia Univ. Press NY.-   Ryan C. A. (1965) Arch Biochem Biophys 110: pp 169.-   Sambrook, J., Fritsch E. F., Maniatis T. (1989) In. Molecular    Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,    Cold Spring Harbor, N.Y.-   Travis J. (1968) Biochem Biophys Res Commun. 30: pp 730.-   Travis J. and Roberts R. C. (1969) Biochemistry 8: pp 2884.-   Trop M. and Birk Y. (1968) Biochem. J. 109: pp 475.-   Wählby S. and Engström L. (1968) Biocheim. Biophys. Acta 151: pp    402.-   Vasquez, J. R. Evnin L. B., Higaki J. N. Craik C. S. (1989) J. Cell.    Biochem. 39: pp. 265.-   Wählby S. (1968) Biochim. Biophys. Acta 151: pp 394.-   Willett, W. S., Gillmor S. A., Perona J. J., Fletterick R. J.,    Craik C. S. (1995) Biochem. 34: pp 2172.-   Yee, L. and Blanch, H. W., (1993) Biotechnol. Bioeng. 41: pp.    781-790.-   WO 97/00316 (NOVO NORDISK AS/Wöldike Helle, Kjeldsen Thomas) A    PROCESS FOR PRODUCING TRYPSIN.-   WO 99/10503 (ROCHE DIAGNOSTICS/Kopetzki Ehrhard, Hopfner Karl-Peter,    Bode Wolfram, Huber Robert) ZYMOGENIC PROTEASE PRECURSOR THAT CAN BE    AUTOCATALYTICALL ACTIVATED AND THEIR USE.-   WO 00/17332 (ELI LILLY AND COMPANY/Hanquier Jose Michael,    Hershberger Charles Lee, Desplancq Dominique, Larson Jeffrey)    PRODUCTION OF SOLUBLE RECOMBINANT TRYPSINOGEN ANALOGS.-   WO 01/55429 (POLYMUN SCIENTIFIC IMMUNBIOLOGISCHE FORSCHUNG    GMBH/Mattanovich Diethard, Katinger Hermann, Hohenblum Hubertus,    Naschberger Stefan, Weik Robert) METHOD FOR THE MANUFACTURE OF    RECOMBINANT TRYPSIN.-   EP 0 597 681 (ELI LILLY AND COMPANY/Greaney Michael Gerard, Rostech    Paul Robert) EXPRESSION VECTORS FOR THE BOVINE TRYPSIN AND    TRYPSINOGEN AND HOST CELLS TRANSFORMED THEREWITH.-   EP 0 699 687 (MITSUBISHI PHARMA CORPORATION/Noda Munehiro, Sumi    Akinori, Ohmura Takao, Yokoyama Kazmasa) PROCESS FOR PURIFYING    RECOMBINANT HUMAN SERUM ALBUMIN.

1. A trypsinogen protein consisting of a first amino acid sequence and asecond amino acid sequence, wherein the C-terminus of the first aminoacid sequence is bound directly to the N-terminus of the second aminoacid sequence, said first amino acid sequence consisting of the aminoacid sequence set forth in SEQ ID NO:26 and said second amino acidsequence consisting of a trypsin amino acid sequence.
 2. The trypsinogenprotein of claim 1 wherein said trypsin amino acid sequence is a porcinetrypsin amino acid sequence.
 3. A trypsinogen protein comprising theamino acid sequence shown in SEQ ID NO:27.
 4. A trypsinogen proteinproduced by the steps comprising a) preparing a yeast host cellcomprising a recombinant nucleic acid that encodes a trypsinogen proteincomprising a signal peptide, the C-terminus of which is bound directlyto the N-terminus of the shortened propeptide amino acid sequence setforth in SEQ ID NO:26, the C-terminus of which is bound directly to theN-terminus of a trypsin amino acid sequence, wherein said signal peptidemediates secretion of trypsinogen from the host cell, b) culturing theyeast host cell in a first culture medium under conditions promoting thegrowth of the yeast host cell; c) culturing the yeast host cell in asecond culture medium having a pH of from 3.0 to 4.0 and inducing theexpression of said recombinant nucleic acid, whereby the yeast host cellexpresses trypsinogen comprising the shortened propeptide in the secondculture medium significantly more than trypsinogen comprising a completepropeptide, and d) isolating the trypsinogen comprising the propeptidefrom the second culture medium.